Cell-specific targeting for delivery of effector moieties such as diagnostic or therapeutic agents is a widely researched field and has led to the development of non-invasive diagnostic and/or therapeutic medical applications. In particular in the field of nuclear medicine procedures and treatments, which employ radioactive materials emitting electromagnetic radiations as γ-rays or particle emitting radiation, selective localization of these radioactive materials in targeted cells or tissues is required to achieve either high signal intensity for visualization of specific tissues, assessing a disease and/or monitoring effects of therapeutic treatments, or high radiation dose, for delivering adequate doses of ionizing radiation to a specified diseased site, without the risk of radiation injury in other e.g. healthy tissues. It is thus of crucial interest to determine and assess cell-specific structures and in particular structures that are present in case of cancer (i.e. tumors) or inflammatory and autoimmune diseases, such as receptors, antigens, haptens and the like which can be specifically targeted by the respective biological vehicles.
The folate receptor (FR) has been identified as one of these structures. The FR is a high-affinity (KD<10−9 M) membrane-associated protein. In normal tissues and organs FR-expression is highly restricted to only a few organs (e.g. kidney, lungs, choroids plexus, and placenta), where it largely occurs at the luminal surface of epithelial cells and is therefore not supplied with folate in the circulation. The FR-alpha is frequently overexpressed on a wide variety of specific cell types, such as epithelial tumours (e.g. ovarian, cervical, endometrial, breast, colorectal, kidney, lung, nasopharyngeal), whereas the FR-beta is frequently overexpressed in leukaemia cells (approx. 70% of acute myelogenous leukaemia (AML) are FR-beta positive). Both may therefore be used as a valuable tumour marker for selective tumour-targeting (Elnakat and Ratnam, Adv. Drug Deliv. Rev. 2004; 56:1067-84). In addition it has recently been discovered that activated (but not resting) synovial macrophages in patients diagnosed with rheumatoid arthritis possess a functionally active FR-beta (Nakashima-Matsushita et al, Arthritis & Rheumatism, 1999, 42(8): 1609-16). Therefore activated macrophages can be selectively targeted with folate conjugates in arthritic joints, a capability that opens possibilities for the diagnosis and treatment of rheumatoid arthritis (Paulos et al, Adv. Drug Deliv. Rev. 2004; 56:1205-17).
Folates is used herein as a generic term for a family of chemically-similar compounds involved in a range of biosynthetic pathways. Folates consist of three units, which include (i) a condensed pyrimidine heterocycle unit, which is linked via a methylene group at the C-6 position to (ii) a p-aminobenzoic acid unit, which is linked to (iii) one or more amino acid units. For example, in the case of folic acid derivatives, a pteridine heterocycle unit is linked via a methylene group at the C-6 position to a p-aminobenzoic acid unit, which is linked to a variable number of glutamic acid units. Each of those three units may be subjected to variation to create a library of various folate structures. Such variations may include folates, that differ in the oxidation state of the pteridine ring, the type of the one carbon substituent at N5 and/or N10 positions, the type and number of conjugated amino acid residues, and the substitution pattern of the various units. Folic acid itself as a synthetic analogue and member of the group of folates is the most oxidized form, whereas dihydrofolate and tetrahydrofolate are progressively more reduced forms of folates (as their name indicates).
Folates are involved in the transfer of 1-C units in key synthetic pathways of bio-molecules such as methionine, purine, and pyrimidine biosynthesis. Additionally, they play an important role in the interconversion of serine and glycine, and in histidine catabolism. Folates and its derivatives have thus been intensively studied over the past 15 years as targeting agents for the delivery of therapeutic and/or diagnostic agents to cell populations bearing folate receptors in order to achieve a selective concentration of therapeutic and/or diagnostic agents in such cells relative to normal cells.
Various probes have been conjugated to folic acid and (pre)clinically evaluated, including folate radiopharmaceuticals (Leamon and Low, Drug Discov. Today 2001; 6:44-51 and Jammaz et al, J. Label Compd Radiopharm 2006; 49:125-137), folate-conjugates of chemotherapeutic agents (Leamon and Reddy, Adv. Drug Deliv. Rev. 2004; 56:1127-41; Leamon et al, Bioconjugate Chem. 2005; 16:803-11), proteins and protein toxins (Ward et al., J. Drug Target. 2000; 8:119-23; Leamon et al, J. Biol. Chem. 1993; 268:24847-54; Leamon and Low, J. Drug Target. 1994; 2:101-12), antisense oligonucleotides (Li et al, Pharm. Res. 1998; 15:1540-45; Zhao and Lee, Adv. Drug Deliv. Rev. 2004; 56:1193-204), liposomes (Lee and Low, Biochim. Biophys. Acta-Biomembr. 1995; 1233:134-44; Gabizon et al, Adv. Drug Deliv. Rev. 2004; 56:1177-92), hapten molecules (Paulos et al, Adv. Drug Deliv. Rev. 2004; 56:1205-17), MRI contrast agents (Konda et al, Magn. Reson. Mat. Phys. Biol. Med. 2001; 12:104-13) etc. Typically all of these probes are conjugated to folic acid through its glutamate portion which lends itself to known carboxylic acid coupling methodology.
Folate radiopharmaceuticals can be in particular very useful for an improved diagnosis and evaluation of the effectiveness of cancer therapy. This may include assessment and/or prediction of a treatment response and consequently improvement of radiation dosimetry. Typical visualization techniques suitable for radio-imaging are known in the art and include positron emission tomography (PET), planar or single photon emission computerized tomography (SPECT) imaging, gamma cameras, scintillation, and the like.
Both PET and SPECT use radiotracers to image, map and measure activities of target sites of choice. Yet, while PET uses positron emitting nuclides which require a nearby cyclotron due to the short half-lives of the positron emitters, SPECT uses single photon emitting nuclides which are available by generator systems, which may make its use independent of nearby facilities such as cyclotrons or reactors and thus more convenient. However SPECT provides less sensitivity than PET and besides a few approaches quantification methods are lacking. In contrast, PET PET shows a higher sensitivity (more than 100-fold of SPECT) and provides well-elaborated quantification methods. Moreover, PET is one of the most sophisticated functional imaging technologies to assess regional uptake and affinity of ligands or metabolic substrates in brain and other organs and thus provides measures of imaging based on metabolic activity. This is for example achieved by administering a positron emitting nuclide to a subject, and as it undergoes radioactive decay the gamma rays resulting from the positron annihilation are detected in the PET scanner by a ring of detectors which are coincidentally connected in pairs.
Factors that need to be considered in the selection of a suitable isotope useful for PET include sufficient half-life of the positron-emitting isotope to permit preparation of a diagnostic composition optionally in a pharmaceutically acceptable carrier prior to administration to the patient, and sufficient remaining half-life to yield sufficient activity to permit extra-corporeal measurement by a PET scan. Furthermore, a suitable isotope should have a sufficiently short half-life to limit patient exposure to unnecessary radiation. Typically, a suitable radiopharmaceutical for PET may be based on a metal isotope, such as gallium or copper. These two require however a chelator for entrapment of the metal, which may have an effect on steric and chemical properties. Alternatively a radiopharmaceutical may be based on a covalently linked isotope which provides minimal structural alteration. Radionuclides used for covalent attachment and suitable for PET scanning are typically positron emitting isotopes with short half lives such as 11C (ca. 20 min), 13N (ca. 10 min), 15O (ca. 2 min) and 18F (ca. 110 min).
To date, a number of chelate-based folate radiopharmaceuticals have been synthesized and successfully evaluated as diagnostic agents for imaging folate receptor-positive tumors. The most widely studied derivatives were labeled either with 111In and 99mTc (Siegel et al., J. Nucl. Med. 2003, 44:700; Müller et al., J. Organomet. Chem. 2004, 689:4712) or with 68Ga (Mathias et al., Nucl. Med. Biol. 2003, 30(7):725). Yet only the latter one is a positron emitter and is suitable for PET imaging while the two former ones are single photon emitters and used for SPECT. Also all of the above need a suitable chelating agent, which is typically linked to folic acid through its amino acid, i.e. glutamate portion.
Thus a folate radiopharmaceutical having a covalently linked positron emitting nuclide would be of great interest. In particular a 18F-labeled folate radiopharmaceutical would be most suitable for PET imaging because of its excellent imaging characteristics which would fulfill all of the above considerations. Compared with other suitable radionuclides (11C, 13N, 15O), 18F is very useful because of its longer half-life of approximately 110 minutes and because it decays by emitting positrons having a low positron energy of 635 keV, which allows a very high-resolution for PET images. Furthermore, the longer half-life of F also allows for syntheses that are more complex and satellite distribution to PET centers with no cyclotron and/or no radiochemistry facilities. In addition the atomic radius of fluorine is comparable to that of H. This implies that steric effects of a fluorine-for-H substitution will hardly interfere with the binding of the ligand to the receptor. Only the high electronegativity of fluorine may influence the biochemical properties of a fluorinated ligand compared to the unsubstituted analogue.
Yet, the structure of folates does not lend itself to direct radiolabeling with 18F. Thus to date, there have been only very few 18F-labeled folates reported in the literature (Bettio et al., J. Nucl. Med., 2006, 47(7), 1153; WO 2006/071754). Moreover, these suggest 18F-labeling through conjugation at the glutamate portion of folates. To date there is no known 18F-labeled folate or derivative thereof, wherein the fluorine-18 is linked within the folate skeleton, such as to the benzoylamine moiety. In addition, the currently reported radiosynthesis was time-consuming and gave only low radiochemical yields of less than 5% (Bettio et al., J. Nucl. Med., 2006, 47(7), 1153) and thus is unsuitable for routine clinical applications.
Thus currently known 18F-labeled folates or derivatives thereof are not able to fill the need for specific radiopharmaceuticals suitable for metabolic imaging of tumors to improve diagnosis and treatment of cancer and inflammatory and autoimmune diseases.
Applicants have now found that 18F-labeled folate radiopharmaceuticals wherein the fluorine-18 is linked to the aminobenzoyl moiety within the folate skeleton may be obtained through for example direct radiolabeling.
Thus, the present invention is directed to new 18F-folate radiopharmaceuticals, wherein the fluorine-18 is covalently linked to the aminobenzoyl moiety which links the pteridine heterocycle to the amino acid portion within folate structures, as well as their precursors, a method of their preparation, preferably through direct radiolabeling, as well as their use in diagnosis and monitoring of cancer or inflammatory and autoimmune disease therapy.